L&#39;intero protocollo dopo l&#39;isolamento delle cellule richiede circa 2 ore. 1. Microsurgical Preparazione <ol><li> Eseguire isolamento delle cellule staminali del midollo osseo del donatore e marcatura fluorescente della sottopopolazione isolato secondo protocolli standardizzati 9,12 permettere alle cellule di riposare durante il tempo viene eseguita l&#39;operazione animale. Per l&#39;etichettatura fluorescente ti consigliamo di CFDA (Carboxyfluorescein diacetate succinimidyl estere). </li><li> Anestetizzare un topo maschio pesa 20-25 g con ketamina (75 mg / kg) e xilazina (2,5 mg / kg). Nel corso degli interventi chirurgici e protocolli sperimentali, anestesia deve essere mantenuta dai integratori (10% di iniezione iniziale, ip) come necessario (di solito ogni 30-60 min). </li><li> Shave delicatamente la faccia anteriore dello scroto destra così come la coscia destra e l&#39;inguine. Raccogliere tutti perdono i capelli con un batuffolo di cotone inumidito. Posizionare il lato ventrale del mouse su un palco di visualizzazione plexiglass e fissare la tassat con telo elastico. Lo stadio è su ordine, costituito da una piastra di base e una seconda piastra sul bordo caudale della piastra di base per l&#39;elevazione di entrambe le gambe e scroto. Posizionare la fase su una piastra elettrica per mantenere la temperatura corporea a 37 ° C. Dopo la fissazione sul palco, mettere l&#39;animale sotto un microscopio di funzionamento ed effettuare le seguenti fasi operative utilizzando 10 - 16 volte di ingrandimento. </li><li> Rendere l&#39;incisione iniziale con le forbici pelle alla coscia destra, solo anteriore dei vasi femorali dal ginocchio fino all&#39;inguine. </li><li> Identificare l&#39;arteria femorale e seguirla prossimale, mobilitando l&#39;arteria da tessuti molli circostanti. Identificare e tagliare un grosso ramo al testicolo sinistro con termocauterio. Successivamente inserire un soggiorno di sutura superficiale al testicolo sinistro. Lieve trazione faciliterà ulteriormente mobilitazione prossimale dell&#39;arteria femorale. </li><li> Dopo il completamento della mobilitazione prossimale, identificare e tagliare un grosso ramo esecuzione medially alla coscia da termocauterio. Successivamente legare l&#39;arteria femorale nella parte distale della coscia destra. Trazione delicata sulla legatura sarà di aiuto ulteriore preparazione. </li><li> Girare l&#39;attenzione ora l&#39;arteria femorale prossimale di nuovo e mettere una sutura intorno all&#39;arteria quanto prossimale possibile. Preparare un nodo, ma non legarlo giù ancora. Applicare una leggera trazione per la sutura e creare un attorcigliamenti arteriosa. </li><li> Dopo aver stabilito il piegamento, incise l&#39;arteria viene accuratamente usando un microscissor. Fate attenzione a non danneggiare la vena femorale. </li><li> Inserire un microcatetere preparata (diametro interno 0,28 millimetri) attraverso l&#39;incisione. Il catetere deve essere anidro (cloruro di sodio 0,9%, NaCl) e collegato ad una siringa da 1 ml prima dell&#39;inserimento. </li><li> Dopo successo inserzione del catetere, allentare attentamente il piegamento per agevolare il passaggio del catetere. Flusso di sangue arterioso dovrebbe essere immediatamente visibile all&#39;interno del catetere. Far avanzare delicatamente il catetere pochi millimetris oltre il piegamento precedente. Successivamente, lega giù la legatura predisposto per il fissaggio del catetere. </li><li> Lavare delicatamente il catetere (0,9% NaCl) e aspirare a confermare la posizione corretta. Usare un pezzo di nastro adesivo per fissare ulteriormente il catetere per la fase di esercizio. </li><li> Dopo la fissazione adeguata, mettere il catetere da parte per le seguenti fasi di preparazione del muscolo cremastere. Applicare lavaggio regolare e l&#39;aspirazione del catetere ogni 5-10 minuti, al fine di prevenire la formazione di coaguli. </li><li> Girare l&#39;attenzione ora allo scroto destra e fare un&#39;incisione tagliando la pelle e la fascia sopra l&#39;aspetto ventrale dello scroto destra. Fare attenzione a non toccare il tessuto sottostante con qualsiasi strumento. </li><li> Estendere l&#39;incisione fino alla piega inguinale e caudalmente all&#39;estremità distale dello scroto. </li><li> Tessuto accuratamente separato sottostante morbido e connettivo sopra il sacco Cremaster, senza toccarlo. Identificare l&#39;estremità distale del Cremaster, e inserire un soggiorno sutura qui per slightly estendere il muscolo. Resecare ora restante del tessuto molle sotto il sacco muscolo tirando delicatamente il muscolo caudale e anteriormente. </li><li> Dopo la separazione completa da tessuto connettivo, aprire il sacco cremastere in un punto sul bordo craniale con le forbici. Estendere l&#39;incisione da questa apertura fino all&#39;estremità distale del muscolo. </li><li> Sigillare la piccola imbarcazione che collega il testicolo e cremastere utilizzando termocauterio, successivamente tagliare i restanti collegamenti tra il muscolo e il testicolo con le forbici. </li><li> Mettere da parte il testicolo, fuori dal campo microscopia. </li><li> Cremastere aperto ora adagiata sul palco. Mettere quattro 6-0 suture Prolene ai bordi del tessuto e fissarli con drappeggio elastico, al fine di diffondere il tessuto del muscolo cremastere. Da ora in poi inumidire continuamente il tessuto con soluzione salina tamponata con fosfato (PBS) usando una siringa da 1 ml (Figura 1). </li><li> Lasciare ora la preparazione riposare per 15 min. Successivamente iniettare0,05-0,1 ml di 1% rodamina-destrano attraverso l&#39;arteria femorale sinistra per migliorare il contrasto del microcircolo. Se si prevedono eventuali chemochine o agenti infiammatori per l&#39;applicazione, si dovrebbe ora topica inumidire il muscolo con il rispettivo reagente con una siringa da 1 ml. Nel caso di un esperimento di controllo, continuare inumidendo con PBS. </li></ol> 2. Intravitale Microscopia <ol><li> Mantenere il mouse fissato sulla fase di esercizio e muoversi sotto un microscopio a fluorescenza modificato per epi-illuminazione e collegato ad un CCD (dispositivo ad accoppiamento di carica) videocamera. Fate attenzione a non dislocare il catetere arteria femorale. </li><li> Condurre esperimenti utilizzando un obiettivo immersione in acqua. </li><li> Dopo aver regolato l&#39;obiettivo per la visualizzazione sufficiente del microcircolo Cremaster e prima iniezione di cellule, stocasticamente definire sei venule postcapillari per una successiva analisi di ferma adesione delle cellule staminali. </li><li> Somministrare cellule staminali fluorescenti marcato,disciolto in 200 microlitri di PBS, a 0.4 x 10 6 cellule per iniezione, con un totale di cinque iniezioni consecutive utilizzando il catetere femorale e una siringa da 1 ml. Agitare con precauzione il campione cellulare prima della prima iniezione. </li><li> Cellule staminali Record rotolamento immediatamente dopo l&#39;iniezione delle cellule, definendo così &quot;rolling&quot; come una riduzione superiore al 50% della velocità cella lungo il rivestimento endoteliale in combinazione con tipico cellulare &quot;bastone e rilasciare&quot; movimenti. Effettuare il conteggio delle cellule staminali di laminazione e di tutte le cellule staminali che passano una distanza venulare predefinita per 1 min di osservazione dopo ogni singola iniezione. La quantità di rotolamento cellule staminali è espressa come percentuale di tutte le cellule che passano. Non contare le cellule espositrici occasionali fenomeni tethering brevi come cellule di rotolamento. </li><li> Dopo l&#39;ultima iniezione cella determinare il numero di cellule staminali saldamente aderenti contando e indica la quantità in relazione alla superficie endoteliale calcolato di the sei venule predefiniti (diametro x lunghezza x π) come cellule aderenti / mm 2. Adesione Studio è considerato quando non il movimento delle cellule è rilevabile per 30 sec. </li><li> Dopo il completamento della microscopia intravitale rimuovere delicatamente il muscolo cremastere e memorizzarlo in corrispondenza successiva analisi (immunoistochimica o biologia molecolare). Sacrifica l&#39;animale da dislocazione cervicale. L&#39;analisi delle registrazioni microscopia intravitale per la quantificazione del gambo rotolamento cellulare e ferma adesione endoteliale, nonché emodinamica microvascolari, come la velocità del sangue rosso cella (mm / sec), la lunghezza della nave (micron), diametro del vaso (micron), parete tasso quota (sec -1); deve essere eseguita in linea da un investigatore cieco al rispettivo trattamento tessutale sperimentale utilizzando un software di elaborazione delle immagini. </li></ol>

<ol>
	<li>Madlambayan, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow+stem+and+progenitor+cell+contribution+to+neovasculogenesis+is+dependent+on+model+system+with+SDF-1+as+a+permissive+trigger.">marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.</a> Blood. 114, 4310-4319 (2009).</li><li>Lapidot, T., Dar, A., Kollet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+stem+cells+find+their+way+home?.">How do stem cells find their way home?.</a> Blood. 106, 1901-1910 (2005).</li><li>Lehr, H. A., Vollmar, B., Vajkoczy, P., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+fluorescence+microscopy+for+the+study+of+leukocyte+interaction+with+platelets+and+endothelial+cells.">Intravital fluorescence microscopy for the study of leukocyte interaction with platelets and endothelial cells.</a> Methods Enzymol. 300, 462-481 (1999).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Leister, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peritoneal+cavity+chamber+for+intravital+microscopy+of+the+liver+under+conditions+of+pneumoperitoneum.">A peritoneal cavity chamber for intravital microscopy of the liver under conditions of pneumoperitoneum.</a> Surg. Endosc. 17, 939-942 (2003).</li><li>Sriramarao, P., Anderson, W., Wolitzky, B. A., Broide, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow-derived+mast+cells+roll+on+P-selectin+under+conditions+of+flow+in+vivo.">Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo.</a> Lab Invest. 74, 634-643 (1996).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cremaster+muscle+preparation+for+intravital+imaging+of+the+microcirculation.">The mouse cremaster muscle preparation for intravital imaging of the microcirculation.</a> J. Vis. Exp. , e2874 (2011).</li><li>Kaminski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+NOS+is+required+for+SDF-1alpha/CXCR4-mediated+peripheral+endothelial+adhesion+of+c-kit++bone+marrow+stem+cells.">Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.</a> Lab Invest. 88, 58-69 (2008).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGB-1+induces+c-kit++cell+microvascular+rolling+and+adhesion+via+both+toll-like+receptor-2+and+toll-like+receptor-4+of+endothelial+cells.">HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.</a> J. Cell Mol. Med. 16, 1094-1105 (2012).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+intravascular+administration+of+mesenchymal+stem+cells+safe?+Mesenchymal+stem+cells+and+intravital+microscopy.">Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy.</a> Microvasc. Res. 77, 370-376 (2009).</li><li>Donndorf, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+migratory+properties+of+human+CD+133(+)+stem+cells+in+vivo+after+intraoperative+sternal+bone+marrow+isolation.">Analysing migratory properties of human CD 133(+) stem cells in vivo after intraoperative sternal bone marrow isolation.</a> Cell Transplant. , (2012).</li></ol>

Tutti gli esperimenti sugli animali, condotti per questo manoscritto sono stati approvati dalla cura degli animali e l&#39;uso comitato locale. Gli autori dichiarano di non avere interessi finanziari in competizione.

<html> Microscopia a fluorescenza intravital permette la visualizzazione diretta e l&#39;analisi quantitativa delle interazioni cellula-endotelio staminali. Il modello cremastere muscolare è particolarmente utile, poiché l&#39;esposizione microchirurgico e le tecniche di visualizzazione intravitale sono stati ben stabiliti nel corso della ricerca sulle interazioni leucociti-endotelio e vari componenti chimici può essere applicato selettivamente al tessuto bersaglio semplice tecnica superfusione. A causa dell&#39;assen...

Lo scopo del presente articolo è quello di descrivere la tecnica della microscopia intravitale (IVM) applicato sul cremastere microcircolazione del mouse per l&#39;osservazione diretta e l&#39;analisi del midollo osseo migrazione di cellule staminali periferiche. Rigenerazione dei tessuti e organi Il concetto attuale di cellule staminali basato comporta la homing delle cellule staminali del midollo osseo al tessuto danneggiato 1. Un passo fondamentale per la migrazione di cellule staminali successo include staminali interazione delle cellule con l&#39;endotelio locale all&#39;interno dell&#39;organo feriti seguita dalla migrazione transendoteliale e, infine, organo attecchimento 2. Analisi intravital di queste cellule staminali - interazioni cellule endoteliali costituisce un parametro ideale per la quantificazione di una risposta rigenerativa basata sulle cellule staminali in differenti impostazioni fisiopatologiche in vivo. Inoltre, sembra concepibile, che, in futuro, analisi intravital della capacità migratoria di specifico p cellule staminaliopulations all&#39;interno di ambienti microcircolazione standardizzati, potrebbero essere applicati prima avanzando queste popolazioni a ulteriori test clinici. Microscopia intravital è stato inizialmente sviluppato per l&#39;osservazione e la quantificazione delle interazioni cellula-endoteliale dei leucociti in vivo nel campo della ricerca infiammatorio 3. Le prime registrazioni di successo di studi di microscopia intravitale sono stati riportati da Cohnheim già nel 19 ° secolo, studiando lingue e mesenteri rana sotto un microscopio ottico 4. Dal suo primo uso IVM ha subito un enorme sviluppo tecnico e oggi alcuni componenti essenziali costituiscono prerequisiti per eseguire IVM quantitativa: (i) preparazioni di tessuto che consentono l&#39;accesso ottico, (ii) sonde molecolari che possono essere rilevati da un microscopio, (iii ) un microscopio collegato ad un sistema di rilevamento e (iv) sistemi di analisi computerizzati in grado di estrarre i parametri di interesse dati di immagineset 4. Una varietà di preparazioni di tessuto è stato introdotto per gli studi IVM compresi i mesenteri e il fegato di topo e nel ratto 5, le camere plica dorsale di topo 6 e il criceto, l&#39;orecchio di coniglio e il sacchetto criceto guancia per citarne alcuni. Tuttavia, nel seguito ci concentreremo sul muscolo cremastere topo, che rappresenta un tessuto ideale per le osservazioni intravitale, come la preparazione e la visualizzazione sono possibili da una procedura chirurgica ben standardizzata, e in generale non si verifichino problemi di artefatti da movimento. La preparazione cremastere muscolare aperta si è svolta per la prima volta nel 1970 da Baez e colleghi 7. Originariamente descritto per i ratti, è stato adottato con successo anche al mouse 8. Dopo gli studi precedenti si erano concentrati principalmente sulle interazioni leucociti con la parete del vaso, il nostro gruppo ha recentemente introdotto il mouse preparazione cremastere muscolo come valutaziostrumento BLE per visualizzazione diretta e l&#39;analisi quantitativa di staminali interazioni cellula-endotelio in un microambiente definito 9. Varie sottopopolazioni di cellule staminali sono state studiate utilizzando questo modello, compresi murino c-kit + cellule staminali del midollo osseo e di cellule staminali mesenchimali, nonché CD umano cellule staminali del midollo osseo 133 + 10-12. Dopo aver isolato cellule da donatore di midollo osseo e marcatura fluorescente per la visualizzazione, le cellule staminali vengono selettivamente applicati nella microcircolazione cremastere utilizzando una iniezione arteriosa attraverso l&#39;arteria femorale, evitando così qualsiasi intrappolamento cella all&#39;interno organi remoti. Inoltre, il modello cremastere muscolare è particolarmente utile in quanto varie chemochine potenzialmente mediano la migrazione delle cellule staminali locale entro le rispettive impostazioni, possono essere applicati localmente al tessuto bersaglio di semplice tecnica superfusione.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Carboxy-fluorescein diacetate succinimidylester (CFDA)</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>C1157</td>
		</tr>
		<tr>
			<td>Rhodamine 6 G (1%)</td>
			<td>Sigma-Aldrich, Munich, Germany</td>
			<td>83697</td>
		</tr>
		<tr>
			<td>Ketamin (Ketanest)</td>
			<td>Pfizer, Berlin, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Xylacin (Rompun)</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate - buffered saline (PBS)</td>
			<td>PAN Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
		</tr>
	</tbody>
</table>

intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration

Nell&#39;era della intravascolari protocolli di applicazione di cellule nel contesto della terapia cellulare rigenerativa, i meccanismi alla base della migrazione di cellule staminali per nonmarrow tessuto non sono stati completamente chiariti. Descriviamo qui la tecnica della microscopia intravitale applicata al cremastere microcircolazione del mouse per l&#39;analisi del midollo osseo periferico migrazione delle cellule staminali in vivo. Microscopia intravitale del cremastere M. è stato precedentemente introdotto nel campo della ricerca infiammatorio per l&#39;osservazione diretta dell&#39;interazione dei leucociti con l&#39;endotelio vascolare. Da staminali periferiche sufficiente e la migrazione delle cellule progenitrici include analoghe fasi iniziali di laminazione lungo e ferma adesione alla endoteliali che rivestono è concepibile applicare il modello cremastere M. per l&#39;osservazione e la quantificazione dell&#39;interazione delle cellule staminali somministrate intravasculary con l&#39;endotelio. Come vari componenti chimici possono essere applicati selettivamente al bersaglio trilascio da semplici tecniche di superfusione, è possibile stabilire condizioni essenziali del microambiente, per l&#39;assunzione iniziale di cellule staminali che si terrà in un organismo vivente al di fuori del midollo osseo.

In generale, l&#39;interazione di direttamente iniettato cellule staminali o progenitrici con l&#39;endotelio vascolare all&#39;interno della microcircolazione cremastere muscolare è un evento raro e avviene esclusivamente in venule postcapillari (diametro: 30-80 micron). A causa fluorescenza etichettatura rotolamento e cellule staminali saldamente aderenti possono essere chiaramente quantificati in separazione da leucociti circolanti endogeni nelle venule osservati (Figura 2). <p class="jove_conte...

Watch this Scientific Journal Video about Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration at JoVE.com

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Microscopia intravitale del microcircolo cremastere topo M. offre un unico e ben standardizzato-modello in vivo per l&#39;analisi del midollo osseo migrazione di cellule staminali periferiche.

Microscopia intravitale del microcircolo nel muscolo mouse Cremaster per l&#39;analisi di migrazione delle cellule staminali periferiche

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

intravital-microscopy-microcirculation-mouse-cremaster-muscle-for

Research

JoVE Journal

Biology

15.1K Views.  University Rostock.  Microscopia intravitale del microcircolo cremastere topo M. offre un unico e ben standardizzato-modello in vivo per l'analisi del midollo osseo migrazione di cellule staminali periferiche. 

Video: Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

La preparazione muscolare mouse Cremaster for Imaging intravitale del microcircolo

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Ricerca

Biologia

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis e Micromanipolazione per intravitale Imaging di fluorescenza del microcircolo

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identificazione dei fattori chiave di regolazione auto-rinnovamento e differenziazione in cellule ematopoietiche precursori EML da Analysis RNA-sequencing

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Visualizzazione e la quantificazione dello strato acellulare nelle arteriole del muscolo Cremaster Rat

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Analisi del trasferimento di gig Junction-dependent di miRNA con la microscopia 3D-FRAP

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

<meta charset="utf8"></head><body>Microscopia subcellulare intravitale della ghiandola mammaria

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

Microscopia subcellulare intravitale della ghiandola mammaria

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

Imaging dal vivo della dinamica dei microtubuli nelle cellule di glioblastoma che invadono il cervello del pesce zebra

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

Methods Article

Microscopia intravitale del microcircolo nel muscolo mouse Cremaster per l'analisi di migrazione delle cellule staminali periferiche